Messenger RNA expression in mammalian cells is highly regulated. Traditionally, emphasis has been placed on elucidating mechanisms by which genes are regulated at the transcriptional level; however, steady-state levels of mRNA is also dependent on its half-life or degradation rate. Changes in mRNA stability play an important role in modulating the level of expression of many eukaryotic genes and different mechanisms have been proposed for the regulation of mRNA turnover (Cleveland and Yen, 1989, New Biol. 1:121; Mitchell and Tollervey, 2000, Curr. Opin. Genet. Dev. 10:193; Mitchell and Tollervey, 2001, Curr. Opin. Cell. Biol. 13:320; Ross, J. 1995, Microbiol. Rev. 59:423; Sachs, A. B., 1993, Cell 74:413; Staton et al. 2000, J. Mol. Endocrinology 25:17; Wilusz et al. 2001, Nat. Rev. Mol. Cell Biol. 2:237). However, the regulation of mRNA stability is complex. Regulation can involve sequence elements in the mRNA itself, activation of nucleases, as well as the involvement of complex signal transduction pathway(s) that ultimately influence trans-acting factors' interaction with mRNA stability sequence determinants.
Recently, it has become increasingly apparent that the regulation of RNA half-life plays a critical role in the tight control of gene expression and that mRNA degradation is a highly controlled process. RNA instability allows for rapid up- or down-regulation of mRNA transcript levels upon changes in transcription rates. A number of critical cellular factors, e.g. transcription factors such as c-myc, or gene products which are involved in the host immune response such as cytokines, are required to be present only transiently to perform their normal functions. Transient stabilisation of the mRNAs which code for these factors permits accumulation and translation of these messages to express the desired cellular factors when required; whereas, under nonstabilised, normal conditions the rapid turnover rates of these mRNAs effectively limit and “switch off’ expression of the cellular factors. Thus, aberrant mRNA turnover usually leads to altered protein levels, which can dramatically modify cellular properties.
The stabilization of mRNA appears to be a major regulatory mechanism involved in the expression of inflammatory cytokines, growth factors, and certain proto-oncogenes. In the diseased state, mRNA half-life and levels of disease-related factors are significantly increased due to mRNA stabilization (Ross, J. 1995, Microbiol. Rev. 59:423; Sachs, A. B., 1993, Cell 74:413; Staton et al. 2000, J. Mol. Endocrinology 25:17; Wilusz et al. 2001, Nat. Rev. Mol. Cell Biol. 2:237). Transcription rates and mRNA stability are often tightly and coordinately regulated for transiently expressed genes such as c-myc and c-fos, and cytokines such as IL-1, IL-2, IL-3, TNFα, and GM-CSF. In addition, abnormal regulation of mRNA stabilisation can lead to unwanted build up of cellular factors leading to undesirable cell transformation, e.g. tumour formation, or inappropriate and tissue damaging inflammatory responses.
Although the mechanisms which control mRNA stability are far from understood, sequence regions have been identified in a number of mRNAs, which appear to confer instability on the mRNAs which contain them. These sequence regions are referred to herein as “mRNA instability sequences”. For example, typical mRNA instability sequences are the AREs (adenylate/uridylate (AU) rich elements), which are found in the 3′ UTR (3′ untranslated region) of certain genes including a number of immediate early genes and genes coding for inflammatory cytokines, e.g. IL-1β and TNFα. The best characterized AU-rich element is the so-called Shaw-Kamen box or AUUUA motif (Shaw and Kamen, 1986, Cell 46:659). Multiple AUUUA sequences (in close proximity or in tandem) or AU-rich regions have been implicated in mRNA instability. For example, mRNA instability sequences described in the literature references identified below contain one or more copies of sequence motifs, e.g. selected from: AUUUA; UAUUUAU; UUAUUUA(U/A)(U/A), and AUUUAUUUA. Typically, in order to function as an instability determinant, the AUUUA motifs should be arranged in tandem, forming at least one UUAUUUAU/AU/A element (Lagnado et al., 1994, Mol. Cell. Biol. 14:7984).
The following publications include extensive discussion of mRNA instability sequences and AREs, the sequences motifs, which they contain and (minimum) sequence requirements for mRNA destabilisation, as well as identifying a number of mRNA instability sequences and the genes which contain them:    Shaw and Kamen, Cell, 1986, 46:659-667 (GM-CSF);    Shyu et al., Genes & Development, 1991, 15:221-231 (c-fos);    Sachs, Cell, 1993, 74:413-421 (Review. “Messenger RNA Degradation in Eukaryotes”);    Chen et al., Mol. Cell. Biol., 1994, 14:416-426 (c-fos);    Akashi et al., Blood, 1994, 83:3182-3187 (GM-CSF etc.);    Nanbu et al., Mol. Cell. Biol., 1994, 14:4920-4920 (uPA);    Stoecklin et al., J. Biol. Chem., 1994, 269:28591-28597 (IL-3);    Lagnado et al., Mol. Cell. Biol., 1994, 14:7984-7995 (general);    Zhang et al., Mol. Cell. Biol., 1995, 15:2231-2244 (yeast);    Zubiaga et al., Mol. Cell. Biol., 1995, 15:2219-2230 (general);    Winstall et al., Mol. Cell. Biol., 1995, 15:3796-3804 (c-fos, GM-CSF);    Chen et al., Mol. Cell. Biol., 1995, 15:5777-5788 (c-fos, GM-CSF);    Chen et al., TIBS, 1995, 20:465-470 (review);    Levy et al., J. Biol. Chem., 1996, 271:2746-2753 (VEGF);    Kastelic et al., Cytokine, 1996, 8:751-761;    Crawford et al., J. Biol. Chem., 1997, 272:21120-21127 (TNFα);    Xu et al., Mol. Cell. Biol., 1997, 18:4611-4621 (general);    Danner et al., J. Biol. Chem., 1998, 273:3223-3229 (human β2-Adrenergic Receptor);    Lewis et al., J. Biol. Chem., 1998, 273:13781-13786 (TNFα);    Chen, C.-Y. and Shyu, A.-B., Mol. Cell. Biol., 1994, 14:8471-8482; and    Klausner, R. et al., Cell, 1993, 72:19-28.
This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present invention. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the present invention.